| Objective:Screening of differentially expressed genes in BMSCs from osteoporosis rats intervened by geraniin through gene chip.the gene overexpression technique was used to establish of overexpression of PPAR? gene in BMSCs from osteoporosis rats and the change expression of key signaling molecules ?-catenin and Runx2 of Wnt/?-catenin signaling pathway.To explore the regulatory effects of geraniin on PPAR?,?-catenin and Runx2,and further explore the molecular mechanism of geraniin in preventing and treating osteoporosis.Methods:1.OVX were performed on 4-month-old female SD rats.Sixteen weeks later,bone mineral density was measured by dual-energy X-ray absorptiometry to determine whether OP rat model was successfully duplicated.2.BMSCs were isolated and cultured in vitro by mechanical methods.The biological characteristics of BMSCs were identified by flow cytometry3.BMSCs were intervened by geraniin(10-9 mol/L)for 14 days,The effects of geraniin on the osteogenic differentiation of BMSCs were observed by Alizarin red staining,and ALP activity assay,respectively.4.After the intervention of geraniin in BMSCs of rats,gene expression profiles of sham-operated BMSCs control group,osteoporotic model BMSCs group and geraniin intervented BMSCs group were detected by chip technology.Compared with model group,genes changed after intervention of geraniin,especially key genes related to osteogenic differentiation of BMSCs,were determined by GO analysis to determine whether these changed genes were enriched on Wnt/?-catenin pathway and verified by real time fluorescence quantitative PCR(RTQ-PCR)5.The down-regulated key genes PPARy were determined in the geraniin-intervented group,and the stablely transferred cell lines were esbablished using lentivirus as vector to carry the PPARy gene to transfect BMSCs through over-expression technology.6.BMSCs transfected with genes were preconditioned for 14 days by geraniin.The effects of geraniin on the protein and mRNA expressions of PPAR?,?-catenin,and Runx2 were determined by use of Western blotting and RTQ-PCR,respectively.Results1.Alizarin red staining showed that the number of calcium nodules in geraniin(10-9mol/L)group increased significantly,similar to the results of ALP activity test Geraniin was further verified to promote osteogenic differentiation of BMSCs.2.PPAR? might be a possible down-regulated target by geraniin according to the gene chip data,the KEGG database,and Pathway analysis.3.The stable cell line of PPAR? overexpression was established through constructing an overexpression vector,transfecting the constructed overexpression vector carrying PPAR y gene into BMSCs.4.The results of Western blotting and RTQ-PCR showed that After PPAR?overexpression,PPAR? expression increased,?-catenin and Runx2 expression decreased(P<0.05).After 10-9 mol/L geranium intervention,PPAR? expression decreased and ?-catenin and Runx2 expression increased(P<0.05).Conclusions1.Geraniin can obviously promote the osteogenic differentiation of BMSCs.2.Over-expression of PPARy can inhibit the expression of ?-catenin and Runx2 in osteoporotic rat BMSCs.3.Geraniin can down-regulate PPAR? protein and mRNA expression and increase the expression of ?-catenin and Runx2 genes4.Geraniin promotes the osteogenic differentiation of BMSCs probably by inhibiting PPAR? signal pathway,upregulating ?-catenin and Runx2,and activating Wnt/?-catenin pathway. |
PDF Full Text Request